Despite more than a century of study the mechanism for slow growth rates in mycobacteria is not understood. A study of mycobacterial regulatory genes led to the isolation of the Mycobacterium smegmatis whmD gene which encodes a homologue of the Streptomyces coelicolor WhiB protein required for early sporulation control. Unlike its Streptomyces homologue, M. smegmatis whmD is an essential gene that could only be insertionally inactivated in the presence of a complementing allele in trans. To examine the phenotype of whmD withdrawal, a conditionally expressing whmD mutant was constructed by fusing the complementing whmD gene to the chemically-regulated acetamidase promoter and regulatory gene block. Upon withdrawal of the inducer acetamide, the conditional whmD mutant exhibited irreversible, filamentous, branched growth with diminished septum formation and aberrant septal placement. Nucleic acid synthesis and levels of the essential cell division protein FtsZ were unaltered by WhmD withdrawal. whmD mRNA and WhmD protein were both stably expressed throughout the growth cycle, but became undetectable in late stationary phase. WhmD overexpression resulted in growth retardation and hyperseptation. Together, these phenotypes indicate a role for WhmD in M. smegmatis septum formation and cell division. A closely related protein, WhmB, which is a homologue of the S. coelicolor WhiD late sporulation regulator, has been shown to be in specifically induced in macrophages in vitro and during granuloma formation in vivo in the Mycobacterium marinum-frog model. Hence this related gene appears to be a non-essential, in-vivo induced virulence gene in certain mycobacteria. In this proposal we will use complete genomic microarrays and real-time RT-PCR to study the pattern of transcriptional regulation of the whiB-like genes of both M. tuberculosis and M. smegmatis. Additionally we will characterize alterations in whm gene expression patterns in mycobacterial mutants lacking key regulatory genes (such as sigma factor genes or other members of the whiB- like family). We will evaluate the consequences of genetic deletion of whmB and whmD in M. tuberculosis and will use the knockout mutants (or conditionally complemented mutants should the gene[s] be essential) in virulence studies in the mouse model. We also propose a characterization of the biochemistry of M. smegmatis WhmD by testing the hypothesis that its 4 cysteines bind a metal ion (such as Zn or Fe), determining which regions of the proteins specify function by genetic domain-swapping experiments and cross-complementation with existing Streptomyces whi mutants, and by seeking proteins which interact with WhmD with the yeast 2-hybrid system. We will also pursue the 3-dimensional structure of the M. smegmatis and M. tb. WhmD proteins and potentially other family members using high resolution NMR. Lastly we will examine the role of WhmD and WhmB in cell physiology by evaluating their sub-cellular localization through GFP tagging and fluorescence microscopy. This latter line of investigation promises to provide important clues as to the roll of WhmD in mycobacterial cell division and the basis for filamentation, branching, and hyposeptation upon genetic withdrawal of whmD expression.